The process of aging is believed to involve progressive loss of control of transcriptional regulation, in particular involving regulatory mechanisms referred to as ?epigenetic?. These changes have been mostly characterized as an increase in variability of DNA methylation with age, referred to as epigenetic drift, with a subset of loci showing an intriguing, progressive change of DNA methylation that appears to act as an ?epigenetic clock?. We note, however, that a DNA methylation assay reports much more than the transcriptional regulatory state of the cells studied. DNA methylation differences between individuals are now appreciated to indicate, for example, cell subtype compositional or DNA sequence differences, without any cells necessarily having changed their transcriptional regulation. DNA methylation is thus both a readout of transcriptional regulation and of other molecular and cellular processes, all generating changes in DNA methylation of the same modest magnitude. To perform a study that allows DNA methylation changes to be interpreted with confidence, we need to understand the sources of variability affecting this transcriptional regulator. A rigorous study should therefore include test genotypes, cell subtype proportions, and transcriptional variability, all of which can change DNA methylation values in a cell population. Furthermore, understanding the cis-regulatory landscape in the cells tested is essential, as this allows a focused analysis at loci informative for DNA methylation changes. An ideal cell type to use in an aging study is CD4+ T lymphocytes. Not only is this a cell type that appears to mediate a number of age-related phenotypes, and is accessible from peripheral blood for genome-wide assays of cohorts, it can also be tested for repertoire diversity using T cell receptor assays, and for cell subtype composition using multiple orthogonal techniques. We will use the strengths of our institution?s Nathan Shock Center of Excellence in the Biology of Aging to collect a well-phenotyped cohort for this study. Our data will reveal whether age-associated epigenetic changes occur independently of confounding influences, but will also allow us to uncover the other cellular and molecular events taking place in CD4+ T cells. An innovative goal is the identification of age-labile functional sequence variants, loci that change their ability to influence gene expression with age, a unique insight into the relationship between DNA sequence polymorphism and aging. We will also test the hypothesis that epigenetic clock CpGs represent loci mediating age-associated glucocorticoid receptor resistance, a separate model for age-associated compromise in cellular function. The outcome of this project will be the most rigorous and definitive study to date of epigenetic changes in aging. We expect human aging to involve a combination of cellular and molecular events. These findings will allow new, comprehensive insights into how CD4+ T cells are involved in mediating age-related diseases.